Hydrophilic metal complexes

ABSTRACT

The present invention concerns new hydrophilic metal complexes and their use as luminescent marker groups in immunoassays.

The present invention concerns new hydrophilic metal complexes and theiruse as luminescent marker groups in immunoassays.

Luminescent metal complexes are known from the state of the art. EP-A-0178 450 discloses ruthenium complexes that are coupled to animmunologically active material in which the ruthenium complexes containthree identical or different bicyclic or polycyclic ligands with atleast two nitrogen-containing heterocycles and at least one of theseligands is substituted with at least one water-solubilizing group suchas --SO₃ H or --COOH and at least one of these ligands is substituteddirectly or via a spacer group with at least one reactive group such as--COOH and the ligands are bound via nitrogen atoms to the ruthenium.

EP-A-0 580 979 discloses the use of osmium or ruthenium complexes asmarker groups for electrochemiluminescence. Heterocycles containingnitrogen such as bipyridines are mentioned as ligands for thesecomplexes. WO 87/06706 discloses further metal complexes which aresuitable as marker groups for electrochemiluminescence measurements.

The disadvantages of the known metal complexes of the state of the artare a poor quantum yield in electrochemiluminescence measurements due tooxygen quenching and photodissociation or/and a high unspecific bindingto proteins.

Therefore the object underlying the present invention was to at leastpartially eliminate the disadvantages of they state of the art.

Surprisingly it was found that the introduction of C₂ -C₃ alkyleneoxy,C₂ -C₃ alkylenethio or/and C₂ -C₃ alkylene-amino units and in particularethylene glycol or/and propylene glycol units into luminescent metalcomplexes reduces the adsorption of conjugates of these complexes withan immunologically reactive substance and thus also improves thestability and recovery of the conjugates in immunoassays. Moreover anincreased quantum yield can be achieved.

In addition it was found that the properties of metal complexes can alsobe improved by introducing polyhydroxy units. These polyhydroxy unitscan be extended to form dendrimer-like structures with severalgenerations. Moreover incorporation of polyamine structures enables theelectron donor required for electrochemiluminescence measurements to beintegrated directly into the ligand sphere of the complex.

A further improvement according to the invention concerns metalcomplexes in the form of a cage or semi-cage in which the ligands arelinked together singly or multiply preferably via hydrophilic spacers.This also leads to a substantial improvement of the photostability andto a reduction of the oxygen quenching.

One subject matter of the present invention is thus a metal complex ofthe general formula (I):

    [M(L.sub.1 L.sub.2 L.sub.3 ].sub.n --X.sub.m A             (I)

in which M is a divalent or trivalent metal cation selected from rareearth or transition metal ions, L₁, L₂ and L₃ are the same or differentand denote ligands with at least two nitrogen-containing heterocycleswherein L₁, L₂ and L₃ are bound to the metal cation via nitrogen atoms,X is a reactive or activatable functional group which is covalentlybound to at least one of the ligands L₁, L₂ and L₃, n is an integer from1 to 10, m is an integer from 1 to 6 and is preferably 1 to 3 and Adenotes one or several negatively charged groups that are required tobalance the charge wherein the complex contains at least one hydrophilicgroup selected from C₂ -C₃ alkyleneoxy units, C₂ -C₃ alkylenethio units,C₂ -C₃ alkyleneamino units and polyhydroxy units.

The metal complex is preferably a luminescent metal complex i.e. a metalcomplex which can generate a detectable luminescence reaction. Thisluminescence reaction can for example be detected by fluorescence or byelectrochemiluminescence measurement. The metal cation in this complexis for example a transition metal or a rare earth metal. The metal ispreferably ruthenium, osmium, rhenium, iridium, rhodium, platinum,indium, palladium, molybdenum, technetium, copper, chromium or tungsten.Ruthenium, iridium, rhenium, chromium and osmium are particularlypreferred. Ruthenium is most preferred.

The ligands L₁, L₂ and L₃ are ligands containing at least twonitrogen-containing heterocycles. Aromatic heterocycles are preferredsuch as bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl. The ligandsL₁, L₂ and L₃ are particularly preferably selected from bipyridine andphenanthroline ring systems.

The reactive or activatable functional group X of the complex is areactive group that can be coupled to an immunological active substanceor an activatable group that can be converted in a simple manner intosuch a reactive group. The group X is preferably an activated carboxylicacid group such as a carboxylic acid halogenide, a carboxylic acidanhydride or an active ester e.g. an N-hydroxysuccinimide ester,p-nitrophenyl ester, pentafluorophenyl ester, imidazolyl ester orN-hydroxybenzotriazolyl ester, a maleimide, an amine,a carboxylic acid,a thiol, a halogenide, a hydroxyl or a group which can bephotoactivated.

In addition the complex contains one or several negatively chargedgroups A required for charge equalization. Examples of suitablenegatively charged groups are halogenides, OH⁻, carbonate, alkylcarboxylate e.g. trifluoroacetate, sulfate, hexafluorophosphate andtetrafluoroborate groups. Hexafluorophosphate, trifluoroacetate andtetrafluoroborate groups are particularly preferred.

The metal complex according to the invention differs from the metalcomplexes known from the state of the art in that it contains at leastone hydrophilic group selected from C₂ -C₃ alkyleneoxy units, C₂ -C₃alkylenethio units, C₂ -C₃ alkyleneamino units and polyhydroxy units.

The polyhydroxy units are preferably selected from groups of formulae(IIa) or (IIb):

    --NR--W                                                    (IIa)

    --O--W                                                     (IIb)

in which W denotes an organic residue with at least two hydroxy groupsand R denotes hydrogen or C₁ -C₅ alkyl, preferably hydrogen or C₁ -C₃alkyl. The organic residue W preferably contains 2 to 6 and particularlypreferably 2 to 4 hydroxy groups. Furthermore W should advantageouslycontain 2 to 10 and in particular 3-6 carbon atoms. Specific examples ofsuitable polyhydroxy units are residues of polyalcohols such as glycerolor aminopolyalcohols. A preferred aminopolyalcohol is Tris(2-amino-2-(hydroxymethyl)-1,3-propanetriol). In this case thepolyhydroxy unit has the formula NH--C(CH₂ OH)₃. The polyalcohols oraminopoly-alcohols are preferably coupled to the metal complex in theform of esters or amides.

The C₂ -C₃ alkyleneoxy, C₂ -C₃ -alkylenethio and C₂ -C₃ -alkyleneaminounits of the metal complex according to the invention are preferably C₂units and in particular ethyleneoxy units. The complex preferablycontains 1 to 30 and particularly preferably 2 to 20 C₂ -C₃ alkyleneoxy,C₂ -C₃ alkylenethio or C₂ -C₃ alkyleneamino units per metal cation.These units are components of substituents of the heterocyclic ligandsof the metal complex. They can be present in the linker between one ofthe ligands and the reactive or activatable functional group X or/and inmonosubstituents. The alkyleneoxy, alkylenethio or alkyleneamino unitscan also be linked together via a bridgehead which can optionally carrya functional group X. On the other hand several complex units can alsobe linked together via the bridgehead. Examples of preferred embodimentsof the metal complexes of the present invention are stated in thefollowing.

In a first embodiment of the present invention the metal complexaccording to the invention has the general ##STR1## in which M, X and Aare defined as above, R₁, R₂, R₃, R₄, R₅ and R₆ are the same ordifferent and each denotes one or several substituents provided that Xis linked to one of the ligands via one of the substituents R₁, R₂, R₃,R₄, R₅ or R₆ and that at least one of the substituents R₁, R₂, R₃, R₄,R₅ or R₆ contains at least one hydrophilic group selected from C₂ -C₃alkyleneoxy, C₂ -C₃ alkylenethio, C₂ -C₃ alkyleneamino units.

The ligands of the complex may also be substituted phenanthroline orbipyridine systems depending on the presence or absence of the groupsindicated by the broken lines.

The substituents R₁, R₂, R₃, R₄, R₅ and R₆ on the ligands are--providedthey do not contain a hydrophilic group--preferably hydrogen, C₁ -C₅alkyl and in particular C_(l) -C₃ alkyl. Overall the hydrophilic groupspreferably contain 1 to 30 and particularly preferably 2 to 20alkyleneoxy, alkylenethio or/and alkyleneamino units, especiallyethyleneoxy units.

The hydrophilic group can be a component of a linker between thefunctional group X capable of coupling and one of the ligands. In thiscase the metal complexes preferably have the general formula (IIIa):##STR2## in which M, X and A are defined as above, R₁, R₂, R₃, R₄ and R₅are defined as above, s is an integer from 0 to 6 and preferably from 1to 4 and Y denotes a hydrophilic linker group with 1 to 10, preferablywith 2 to 6 hydrophilic units selected from C₂ -C₃ alkyleneoxy units, C₂-C₃ alkylenethio units, C₂ -C₃ alkyleneamino units, in particularethyleneoxy units.

However, the functional group X does not have to be linked to the ligandvia a hydrophilic linker. In this case the metal complex according tothe invention preferably has the general formula (IIIb): ##STR3## inwhich M, X and A are defined as above, R₁, R₂, R₃, R₄ and R₅ are definedas above, provided that R₁, R₂, R₃, R₄ or/and R₅ contains a hydrophilicsubstituent group which each comprise 1 to 10, preferably 2 to 6 C₂ -C₃alkyleneoxy units, C₂ -C₃ alkylenethio units or/and C₂ -C₃ alkyleneaminounits, in particular ethyleneoxy units.

An example of a compounds of formula (IIIa) is shown in FIG. 1a and 1b.These compounds contain the hydrophilicity only in the linker betweenthe group X--a maleimide (FIG 1a) or an N-hydroxysuccinimide ester (FIG.1b)--and a ligand. The other ligands can however likewise havehydrophilic substituents. An example of a compound of formula (IIIb) isshown in FIG. 1b. In this case the group X is an N-hydroxysuccinimideester.

The ligands of the metal complex according to the invention can also belinked together so that the metal complex is present in the form of asemicage or cage. A preferred embodiment of a metal complex according tothe invention in the form of a semicage or cage has the general formula(IV): ##STR4## in which M, X, n and A are defined as above, R₁, R₂ andR₃ are the same or different and each denote one or severalsubstituents--as defined above--on the bipyridine or phenanthrolineligand and Y in each case denotes a hydrophilic linker group whichcomprises 1 to 10 hydrophilic units selected from C₂ -C₃ alkyleneoxy, C₂-C₃ alkylenethio and C₂ -C₃ alkyleneamino units, in particularethyleneoxy units.

If the substituents R₁, R₂ and R₃ in formula (IV) are covalently linkedtogether optionally via hydrophilic linker groups, then the complex offormula (IV) is in the form of a cage.

Examples of complexes of formula (IV) in a semicage form are shown inFIGS. 2 and 3a. An example of a complex in a cage form is shown in FIG.3b. The group X in FIG. 2 is a carboxyl residue. The metal cation andthe anions are not shown in FIGS. 3a and 3b.

The complex of formula (IV) may not only be present as a monomer but asan oligomer composed of preferably up to 5 individual metal complexes.In this case the functional group X capable of coupling can for examplebe a substituent on an aromatic nucleus e.g. a phenyl nucleus in whichcase two or several of the remaining substituent positions of thearomatic nucleus can be substituted by a metal complex in the form of asemicage or cage.

Examples of oligomeric metal complexes of formula (IV) are shown inFIGS. 4 and 5. The metal ions and anions are not shown in these figures.

In a further preferred embodiment of the present invention the metalcomplex is substituted with polyhydroxy units and has the generalformula (V): ##STR5## in which M, X and A are defined as above, Zdenotes a linker group, R'₁, R'₂, R'₃, R'₄ and R'₅ are the same ordifferent and each denotes one or several substituents e.g. hydrogen orC₁ -C₅ alkyl, in particular C₁ -C₃ alkyl and s is an integer from 0 to6, preferably of 1 to 4 provided that R'₁, R'₂, R'₃ or/and R'₄ contain ahydrophilic substituent group which comprises a polyhydroxy unit.

The ligand X of the metal complex (V) can be coupled to the ligand via ahydrophilic linker e.g. a linker according to formula (IIIa) and alsovia a linker according to formula (IIIb). The substituent R'₅ ispreferably hydrogen or a C₁ -C₅ alkyl group and in particular a C₁ -C₃alkyl group.

An example of a compound of formula (V) is shown in FIG. 6. The group Xis a carboxyl residue.

The OH groups of the polyhydroxy units of metal complexes of the generalformula (V) are optionally substituted by hydrophilic groups e.g. by C₂-C₃ alkylenoxy; C₂ -C₃ alkylenethio units or/and C₂ -C₃ alkyleneaminounits.

In a specific embodiment of the present invention the hydrophilicsubstituent groups of the OH groups of the polyhydroxy units aredendrimers of the general formula (VIa) or (VIb):

    --A.sub.1 --NR--W.sub.1 (A.sub.2 --NH--W.sub.2).sub.n'     (VIa)

    --A.sub.1 --O--W.sub.1 (A.sub.2 --O--W.sub.2).sub.n'       (VIb)

in which

A₁ and A₂ are the same or different and denote linker groups,

W₁ and W₂ are the same or different and denote an organic residue withat least 2 hydroxy groups,

R denotes hydrogen or C₁ -C₅ alkyl and preferably hydrogen or C₁ -C₃alkyl and

n' is 0 or corresponds to the number of hydroxy groups of W₁.

The linker groups A₁ and A₂ are preferably groups of formula (CH₂)_(m)'C(═O)--, in which m' is 1 to 5 and in particular 1 to 3.

The groups W₁ and W₂ are preferably polyhydroxy units which are definedas for the groups of formulae (IIa), (IIb). If n' is zero, then adendrimer of the first generation is present. If n' corresponds to thenumber of hydroxy groups of W₁ a dendrimer of the second generation ispresent. The hydroxy terminal groups of the dendrimers can be optionallysubstituted e.g. by a linker group having the formula A₃ --R' in whichA₃ is defined like the linker groups A₁ and A₂ and R' denotes C₁ -C₅alkyl and preferably C₁ -C₃ alkyl.

The metal complexes according to the invention are produced by reactinga metal salt e.g. a metal halogenide with the appropriate ligands andoptionally subsequently replacing the halogenide ion byhexafluorophosphate or tetrafluoroborate anions. Such processes aredescribed in the state of the art e.g. in EP-B-0 178 450 and EP-B-0 255534. Reference is hereby made to this disclosure.

The production of hydrophilic N-heterocyclic ligands can be carried outin a simple manner by substitution on the aromatic ligand e.g. via atosylate. The hydrophilic linker that carries the functional group X canalso be coupled in a corresponding manner.

The production of metal complexes of formula (IV) with a semicage orcage structure can for example be carried out by attaching alkyleneoxy,alkylenethio or/and alkyleneamino units to the bipyridine orphenanthroline ligands and linking these units to a bridgehead via anether or an amide bond. If two bridgeheads are used it is possible toobtain cage structures. The linking of three ligands to a trivalentbridgehead e.g. Tris is preferred. The complex itself is produced byreaction with metal salts as described above.

The production of metal complexes in a cage or semicage form can beachieved according to reaction scheme III (FIGS. 9a and 9b).

The metal complexes of the general formula (V) are for example producedby a reaction according to scheme I (FIG. 7) in which an appropriatelysubstituted ligand is reacted with an amino polyalcohol or a partiallyprotected polyalcohol in which hydrophilic groups of formulae (IIa) or(IIb) are attached to the ligand.

Dendritical metal complexes can be produced according to reaction schemeII (FIG. 8).

A further subject matter of the present invention is a conjugatecomprising a biological substance to which at least one metal complexaccording to the invention is coupled. Examples of suitable biologicalsubstances are cells, viruses, subcellular particles, proteins,lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids,oligosaccharides, polysaccharides, lipopolysaccharides, cellularmetabolites, haptens, hormones, pharmacologically active substances,alkaloids, steroids, vitamins, amino acids and sugars.

The metal complex is preferably coupled to the biologically activesubstance via the reactive or activatable functional group of the metalcomplex which can covalently couple to a functional group of thebiological substance. If the functional group is an active ester, it canfor example be coupled to the free amino groups of the biologicalsubstance. If the functional group is a maleinimide residue, it can becoupled to the free SH groups of the biological substance. Functionalgroups of the biological substance can also be activated in a similarmanner which can subsequently react for example with a free carboxylicacid, amino or thiol group of the metal complex.

In a particularly preferred embodiment of the present invention themetal complexes are coupled to a peptide which preferably has a maximumlength of 50 amino acids and particularly preferably of 30 amino acids.The production of these peptides labelled with a metal complex ispreferably carried out by synthesizing a peptide with the desired aminoacid sequence on a solid phase in which a) after the synthesis anactivated metal complex, preferably a metal complex-active esterderivative, is coupled to the N-terminal amino group of the peptideor/and b) during the synthesis an amino acid derivative that iscovalently coupled to a metal complex is introduced in at least oneposition of the peptide. The coupling of the metal complex to theN-terminal amino acid of the peptide is preferably carried out beforecleaving the peptide from the solid phase and before cleaving protectinggroups on reactive side groups of the amino acid derivatives used forthe peptide synthesis.

The peptides preferably contain an immunologically reactive epitoperegion and a spacer region wherein at least one metal complex markergroup is coupled to the spacer region. The spacer region preferably hasa length of 1 to 10 amino acids and is located at the amino or/andcarboxy terminus of the peptide.

The spacer region preferably contains amino acids which have chargesor/and can form hydrogen bridges. The amino acids of the spacer regionare preferably formed from the group comprising glycine, β-alanine,γ-aminobutyric acid, ε-aminocaproic acid, lysine and compounds of thestructural formula NH₂ --[(CH₂)_(y) O]_(x) --CH₂ --CH₂ --COOH in which yis 2 or 3 and x is 1 to 10.

The epitope regions of the peptides are preferably derived frompathogenic organisms e.g. bacteria, viruses and protozoa or fromautoimmune antigens. The epitope region is particularly preferablyderived from viral antigens e.g. the amino acid sequences of HIVI, HIVIIor hepatitis C virus (HCV).

Further preferred examples of biological substances are biotin, nucleicacids, antibodies or antibody fragments, polypeptide antigens i.e.immunologically reactive polypeptides or haptens i.e. organic moleculeswith a molecular weight of 150 to 2000, in particular molecules with asteroid backbone such as cardenolides, cardenolide-glycosides (e.g.digoxin, digoxigenin), steroid alkaloids, sexual hormones (e.g.progesterone), glucocorticoids etc. Further examples of haptens areprostaglandins, leuco-en-diines, thromboxanes, pharmacologically activesubstances etc.

Yet a further subject matter of the present invention is the use of themetal complexes according to the invention or of the conjugatesaccording to the invention in an immunological detection method.

In this method the metal complex is used as a marker group with the aidof which it is possible to qualitatively or/and quantitatively determinean analyte in a sample solution. The metal complex is preferablydetected by electrochemiluminescence in which case luminescent speciesare generated electrochemically at the surface of an electrode. Examplesfor carrying out luminescence assays using metal complexes of the stateof the art may be found in EP-A-0 580 979, WO 90/05301, WO 90/11511 andWO 92/14138. Reference is hereby made to the methods and devices forluminescence assays disclosed therein. Electrochemiluminescence assaysare carried out in the presence of a solid phase which is preferablycomposed of microparticles and in particular of magnetic microparticleswhich are provided with a reactive coating e.g. with streptavidin. Inthis manner it is possible to detect immune complexes containing a metalcomplex as a marker group that are bound to a solid phase.

The electrochemiluminescence measurement is preferably carried out inthe presence of a reducing agent for the metal complex e.g an amine.Aliphatic amines and in particular primary, secondary and tertiaryalkylamines, the alkyl groups of which each have 1 to 3 carbon atoms arepreferred. Tripropylamine is particularly preferred. The amine can,however, also be an aromatic amine such as aniline or a heterocyclicamine. The reducing agent can already be integrated into the ligandsphere of the complex. Such systems are especially suitable for thedetermination of analytes that are present in a highly concentratedform.

In addition a non-ionic surface-active agent may also be present as anamplifier e.g. an ethoxylated phenol. Such substances are for examplecommercially available under the names Triton X100 or Triton N401.

On the other hand the luminescent metal complex can also be detected byfluorescence in which case the metal chelate is excited by irradiationwith light of a suitable wavelength and the resulting fluorescentradiation is measured. Examples for carrying out fluorescence assays maybe found in EP-A-0 178 450 and EP-A-0 255 534. Reference is hereby madeto this disclosure.

The present invention is further elucidated by the following examplesand figures.

FIG. 1a shows a metal complex of formula (IIIa),

FIG. 1b shows a metal complex of formula (IIIa),

FIG. 1c shows a metal complex of formula (IIIb),

FIG. 2 shows a metal complex of formula (IV),

FIG. 3a shows a metal complex of formula (IV),

FIG. 3b shows a metal complex of formula (IV),

FIG. 4 shows a metal complex of formula (IV),

FIG. 5 shows a metal complex of formula (IV),

FIG. 6 shows a metal complex of formula (V),

FIG. 7 shows a reaction scheme for the production of metal complexes offormula (V),

FIG. 8 shows a further reaction scheme for the production of metalcomplexes of formula (V),

FIGS. 9a and 9b show a reaction scheme for the production of metalcomplexes of formula (IV),and

FIG. 10 shows a metal complex-progesterone conjugate.

EXAMPLE 1

Production of a hydrophilic bipyridine ligand(4,4'-bis(methoxy-ethoxy-ethoxy)-bipyridine)

50 ml of a solution of lithium diisopropylamide in a mixture ofcyclohexane, ethylbenzene and THF is cooled to -78° C. 350 ml of asolution of 50 mml bipyridine in THF is added dropwise. It is allowed tostir for two hours and a solution of 100 mmolmethoxy-ethoxy-ethoxy-toxylate in THF is added dropwise. After 1 hour at-78° C. the reaction mixture is allowed to stand overnight at roomtemperature. Then an aqueous sodium chloride solution is added.Subsequently the THF is removed using a rotary evaporator and theresidue is extracted with ethyl acetate.

The product is purified chromatographically on silica gel. Eluant: ethylacetate-methanol-ammonia 95/4/1 or amino silica gel using ethylacetate-petroleum ether as the eluant.

H-NMR(CDCl₃): 3.6 ppm (m.CH₂ CH₂)=16H 7.12-8.5 ppm (bpy)=6H

EXAMPLE 2

Production of a bis(bis-ethylene glycol-bipyridine)dichloro-rutheniumcomplex

Ruthenium trichloride is dissolved with a double-molar excess of theligand synthesized in example 1 and a 7- to 8-fold excess of lithiumchloride in DMF and boiled for 6 hours under reflux. The solvent isremoved, the residue is dissolved in water and extracted with ethylacetate and subsequently with chloroform. The chloroform phases arepooled, dried, filtered and concentrated on a rotary evaporator.

The product is purified by thin layer chromatography on amino silica gelusing acetonitrile/H₂ O 10/1 (Rf=0.58).

EXAMPLE 3

Synthesis of bis(bis-ethyleneglycol-bipyridine)-4(4(4'-methyl-2,2'bipyridyl))-butanoic acid

3.0 g of the ruthenium complex synthesized in example 2 was dissolvedunder argon in 240 ml of an ethanol-water mixture. 0.82 gbipyridyl-butanoic acid derivative is added and heated for three hoursunder reflux. The solution is concentrated, washed with ethyl acetateand extracted with chloroform. It is rotary evaporated and the residueis purified on SP-Sephadex (eluant: NaCl/HCl in water).

Yield: 500 mg, purity (HPLC): 93%

MS (PosLIMS): 1455.5=Ru²⁺ complex PF₆ ⁻

EXAMPLE 4

Synthesis of a hydrophilic metal complex active ester derivative

260 mg of the complex synthesized in example 3 was dissolved inmethylene chloride and admixed with an equimolar amount ofdicyclohexylcarbodiimide/N-hydroxysuccinimide ester. It is allowed tostir for twelve hours, the DCH is removed by filtration and it is rotaryevaporated. The crude product is purified by preparative HPLC. The yieldis 85%.

EXAMPLE 5

Synthesis of a hydrophilic metal complex maleimide derivative

150 g of the metal complex Ru(bipyridine)₂(bipyridine-CO-N-hydroxysuccinimide ester) according to EP-A-0 580 979are reacted for ca. 12 hours together with 100 mgmaleimido-amino-dioxaoctane (MADOO) and triethylamine in methylenechloride. The reaction mixture is shaken out three times with water andthe residue from the organic phase is purified on a Sephadex-LH20 columnusing methylene chloride/methanol. The compound Ru(bpy)₂ (bpy-CO-MADOO)shown in FIG. 1a is obtained.

MS: M⁺ =1025.3 (corresponds to Ru²⁺ PF₆ ⁻ complex).

EXAMPLE 6

Preparation of a hydrophilic metal complex active ester derivative

0.5 mmol of the ruthenium complex used as a starting material in example5 in 20 ml dichloromethane is reacted with 0.5 mmolmono-Boc-diaminodioxaoctane in 20 ml dichloromethane and one equivalentof triethylamine. The purification is carried out as described inexample 5. The Boc protecting group is cleaved according to standardmethods (trifluoroacetic acid/methylene chloride).

The resulting product is reacted for 2 h at room temperature with anequimolar amount of suberic acid-bis-N-hydroxysuccinimide ester indimethylformamide. The solvent is removed, the residue is taken up inwater and lyophilized. The resulting product is shown in FIG. 1b.

H-NMR: 7.2-8.9 ppm: bipyridine (22H); 2.8 ppm NHS ester (4H).

EXAMPLE 7

Preparation of a metal-complex-hapten conjugate

10 mg of the N-hydroxysuccinimide ester from example 4 is dissolvedtogether with 3.2 mgprogesterone-3-carboxymethyl-oxime-diamondioxyoctane in 2 ml methylenechloride, 1.2 μl triethylamine is added and it is stirred for 12 hoursat room temperature. The solvent is removed and the residue is purifiedon Sephadex. The resulting conjugate is shown in FIG. 10.

MS (posLIMS): M+=1923.0 (ruthenium-complex-progesterone conjugate²⁺trifluoroacetate)

EXAMPLE 8

Production of metal chelate labelled peptides

The metal chelate labelled peptides were produced by means offluorenylmethyloxycarbonyl-(Fmoc)-solid phase peptide synthesis on abatch peptide synthesizer e.g. from Applied Biosystems A431 or A433. Forthis 4.0 equivalents of the amino acid derivatives shown in Table 1 wereused in each case.

                  TABLE 1                                                         ______________________________________                                        A             Fmoc--Ala--OH                                                     C Fmoc--Cys(Trt)--OH                                                          D Fmoc--Asp(OtBu)--OH                                                         E Fmoc--Glu(OtBu)--OH                                                         F Fmoc--Phe--OH                                                               G Fmoc--Gly--OH                                                               H Fmoc--His(Trt)--OH                                                          I Fmoc--Ile--OH                                                               K1 Fmoc--Lys(Boc)--OH                                                         K2 Boc--Lys(Fmoc)--OH                                                         K3 Fmoc--Lys(BPRu)--OH                                                        L Fmoc--Leu--OH                                                               M Fmoc--Met--OH                                                               N Fmoc--Asn(Trt)--OH                                                          P Fmoc--Pro--OH                                                               Q Fmoc--Gln(Trt)--OH                                                          R Fmoc--Arg(Pmc) --OH                                                         S Fmoc--Ser(tBu)--OH                                                          T Fmoc--Thr(tBu)--OH                                                          U Fmoc--βalanine--OH                                                     V Fmoc--Val--OH                                                               W Fmoc--Trp--OH                                                               Y Fmoc--Tyr(tBu)--OH                                                          Z Fmoc--ε-alminocaproic acid--OH                                      Nle Fmoc--ε-norleucine--OH                                            Abu Fmoc--γ-alminobutyric acid--OH                                    ______________________________________                                    

In the variant (a)--introduction of the metal complex after completionof the solid phase synthesis--an activated hydrophilicruthenium(bipyridyl)₃ complex (BPRu) was coupled to the N-terminal aminoacid of the peptide. The lysine derivative K1 was used for the spacerregion and the lysine derivative K2 was used for the epitope regions

According to variant (b) metal chelate groups were introduced into thepeptide sequence by direct incorporation of metal chelate-coupled aminoacid derivatives e.g. within the sequence via a lysine residueε-derivatized with a metal chelate active ester e.g. the lysinederivative K3 or N-terminally using an α-derivatized amino acid residue.

The amino acids or amino acid derivatives were dissolved inN-methylpyrrolidone. The peptide was synthesized on 400-500 mg4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (TetrahedronLetters 28 (1987), 2107) loaded with 0.4-0.7 mmol/g (JACS 95 (1973),1328). The coupling reactions were carried out for 20 min. with 4equivalents dicyclohexylcarbodiimide and 4 equivalentsN-hydroxybenzotriazole with respect to the Fmoc-amino acid derivative indimethylformamide as the reaction medium. The Fmoc group was cleavedwithin 20 min. after each synthesis step using 20% piperidine indimethylformamide.

When cysteine residues were present in the peptide sequence, the solidphase was oxidized immediately after completing the synthesis usingiodine in hexafluoroisopropanol/dichloromethane.

The peptide was released from the support and the acid-labile protectinggroups were cleaved using 20 ml trifluoroacetic acid, 0.5 mlethanedithiol, 1 ml thioanisole, 1.5 g phenol and 1 ml water in 40 min.at room temperature. The reaction solution was subsequently admixed with300 ml cooled diisopropyl ether and kept at 0° C. for 40 min. untilcomplete precipitation of the peptide. The precipitate was filtered,rewashed with diisopropyl ether, dissolved in a small amount of 50%acetic acid and lyophilized. The crude material obtained was purified bymeans of preparative HPLC on Delta-PAK RP C18 material (column 50×300mm, 100 Å, 15 μ) over a corresponding gradient (eluant A: water, 0.1%trifluoroacetic acid, eluant: B acetonitrile, 0.1% trifluoroacetic acid)in ca. 120 min. The identity of the eluted material was checked by meansof ion spray mass spectrometry.

The metal chelate label was introduced on the free N-terminal aminogroup of the support-bound peptide according to variant (a) viaappropriate active ester derivatives. For this 4 equivalents of ahydrophilic ruthenium (bipyridyl)₃ complex (BPRu) per free primary aminofunction were activated withN-hydroxybenzotriazole/di-cyclohexylcarbodiimide, dissolved in a smallamount of DMSO, added dropwise and stirred at room temperature. Thereaction was monitored by means of analytical HPLC. After cleaving fromthe support, the product was purified by preparative HPLC. The identityof the eluted material was checked by means of ion spray massspectrometry.

The peptides were synthesized by a combination of variants (a) and (b)i.e. incorporation of metal-chelate-coupled amino acid derivativeswithin the sequence, cleaving the N-terminal Fmoc group and reacting thefree N-terminal amino group with a metal-chelate active esterderivative.

When the metal chelate-coupled amino acid derivatives were exclusivelyincorporated directly during the solid phase synthesis according tovariant (b), it was no longer necessary to subsequently introduce metalchelate active esters.

The peptide-metal-complex conjugates shown in Table 2 were prepared fromthe regions gp120, gp41 and gp32 of HIV I and HIV II.

                  TABLE 2                                                         ______________________________________                                        Ruthenylated linear peptides                                                  ______________________________________                                        gpl20 SEQ     BPRu-UZU-NNTRKSISIGPGRAFYT                                        ID                                                                            NO:1                                                                           - SEQBPRU-UZ-NTTRSISIGPGRAFY                                                 ID                                                                            NO:2                                                                           - SEQBPRu-UZ-NTTRSISIGPGRAFY                                                 ID                                                                            NO:2                                                                           - SEQNNTRKSISIGPGRAFYT-K(BPRu)                                               ID                                                                            NO:1                                                                           - SEQBPRU-UZU-IDIQEERRMRIGPGMAWYS                                            ID                                                                            NO:3                                                                           - gp4l/1SEQBPRu-UZU-AVERYLKDQQLLGIW                                          ID                                                                            NO:4                                                                           - SEQBPRu-UGGG-QARILAVERYLKDQQLLGIWGASG                                      ID                                                                            NO:5                                                                           - SEQBPRu-GGGG-QARILAVERYLKDQQLLGIWGASG                                      ID                                                                            NO:5                                                                           - SEQBPRu-UZU-WGIRQLRARLLALETLLQN                                            ID                                                                            NO:6                                                                           - gp4l/2SEQBPRu-UZU-LGIWGCSGKLICTTAV                                         ID                                                                            NO:7                                                                           - SEQBPRu-UGGG-GCSGKLICTTAVPWNASWS                                           ID                                                                            NO:8                                                                           - SEQ(GCSGKLICTTAVPWNASWS)K-(BPRu)                                           ID                                                                            NO:8                                                                           - gp4l/3SEQBPRu-UZU-KDQQLLGIWGSSGKL                                          ID                                                                            NO:9                                                                           - gp4l/4SEQBPRu-UZU-ALETLLQNQLLSLW                                           ID                                                                            NO:10                                                                          - gp32SEQBPRu-UZU-NSWGCAFRQVCHTT                                             ID                                                                            NO:11                                                                          - SEQBPRu-GGG-QAQLNSWGCAFRQVCHTTVPWPNDSLT                                    ID                                                                            NO:12                                                                       ______________________________________                                    

The peptides shown in the following Table 3 were synthesized from theNS5 region, the NS4 region and the Core region of HCV.

                                      TABLE 3                                     __________________________________________________________________________    Ruthenylated linear peptide                                                   __________________________________________________________________________    Corel  SEQ ID NO:13                                                                          BPRU-GGGG-KNKRNTNRR                                               - Corel+2 SEQ ID NO:14 BPRu-UZU-KNKRNTNRRPQDVKFPGGGQIVGGV                     - NS4/1+2 SEQ ID NO:15 BPRu-UZ-SQHLPYIEQG-NleNle-LAEQFKQQALGLLQT                                                      - NS4/3m  SEQ ID NO:16                                                      BPRu-UZ-SRGNHVSPTHYVPESDAA                                                      - NS5/1  SEQ ID NO:17 BPRu-UZ-S                                             RRFAQALPVWARPD                          - Core1+2+3 SEQ ID NO:18 BPRu-UZ-KNKRNTNRRPQDVKFPGGGQIVGGVLLPRR                                                       - Core1m  SEQ ID NO:19                                                      BPRu-UZ-NPKPQKKNKRNTNRR                 - Core3m  SEQ ID NO:20 BPRu-UZ-GQIVGGVYLLPRRGPRLG                             - Core2m       SEQ ID NO:21 BPRu-UZ-PQDVKFPGGGQIVGGV                          - NS4/3m-I    SEQ ID NO:22 BPRu-UZU-SRGNHVSPTHYVPESDAA                        - NS4/1   SEQ ID NO:23 BPRu-UZU-SQHLPYIEQ                                  __________________________________________________________________________

Biotin-labelled peptides were either synthesized N-terminally byderivatization on a resin (biotin active ester) or in the sequence usinga biotin-active ester ε-derivatized lysine residue (Fmoc-Lys (biotin)--OH).

EXAMPLE 9

Preparation of diethyl-α,α,α',α'-tetrakis(ethoxycarbonyl)-2,2'-bipyridine-4,4'-diyl-dipropionate (correspondingto compound (1) in FIG. 7)

2.00 g (5.8 mmol) 4,4'-bis(bromomethyl)-2,2'bipyridine in 40 mltoluene/DMF (3/2) is added dropwise at 50° C. to a mixture of 6.85 g(29.5 mmol) triethyl-methane-tricarboxylate and 4.1 g (29.7 mmol)potassium carbonate in 50 ml toluene/DMF (3/2) while stirringthoroughly. It is stirred for a further 4 days at 65° C., it is filteredand subsequently the solvent is removed in a vacuum. The oily residue istaken up in 100 ml toluene and successively shaken out three times withwater, three times with 7% sodium hydroxide solution and three timeswith water. The organic phase is pooled and dried over sodium sulfate.After removing the volatile components in a vacuum, the residue isrecrystallized from cyclohexane. In order to completely removeimpurities it is separated by column chromatography (SiO₂, CHCl₃ /MeOH(10:1) first band).

Colourless crystals (cyclohexane)

Yield: 2.95 g (79%)

Melting point: 116° C.

¹ H-NMR (250 MHz, CDCl₃, 25° C.): δ=1.21 (t, 18H, ³ J=7.2 Hz), 3.57 (s,4H), 4.22 (q, 12H, ³ J=7.2 Hz), 7.25 (dd, 2H, ³ J=5.1 Hz, ⁴ J=1.2 Hz),8.29 (d, 2H, ⁴ J=1.2 Hz), 8.51 (dd, 2H, ³ J=5.1 Hz, ⁴ J=1.2 Hz)

¹³ C-NMR (75 MHz, CDCl₃, 25° C.): δ=13.96 (CH₃), 38.1 (CH₂), 62.6 (H₂CO), 66.5 (CCO), 123.4, 123.6, 145.9, 149.0, 155 (pyridine-C, CH), 166.5(C═O).

IR (KBr/solid) [cm⁻¹ ]: 556, 610, 863, 1026, 1186, 1258, 1305, 1594,1737 vs. 2988

MS-50: (180° C., 70 eV, 300 μA, m/e): found: 644,2585

C₃₂ H₄₀ N₂ O₁₂ (644,682)

EXAMPLE 10

Preparation ofN,N'-bis(2-hydroxy-1,1-bis(hydroxymethyl)-ethyl)-ethyl-α,α,-bishydroxy-2,2'-bipyridine-4,4'-diyldipropionamide(corresponding to compound (2) in FIG. 7)

967.5 mg (7.00 mmol) potassium carbonate is added while stirring to asolution of 752.7 mg (1.17 mmol) of the hexaester (1) of example 7 and848.6 mg (7.00 mmol) α,α,α-tris-(hydroxy-methyl)-methylamine in 10 mlDMSO dried over CaH₂. After addition of the base the mixture becomesslightly yellow. After stirring for a further 10 minutes at 25° C. thesuspension is centrifuged and the solution is decanted from the solidpotassium carbonate. The solvent is removed in a vacuum (0.001 mbar) at30° C. The yellow, oily residue is suspended in a small amount of waterand the product is precipitated by the slow addition of dry acetone(distilled over P₄ O₁₀). It is completely precipitated in the cold. Thesolution is decanted and the residue is dried for several days over P₄O₁₀. A hygroscopic colourless solid remains which is used withoutfurther purification.

Yield: 0.652 g (67%)

¹ H-NMR (250 MHz, DMSO-d₆, 25° C.): δ=3.08 (d, 4H), 3.22 (s, 8H), 3.47(d, 8H, ² J=10.8 Hz), 3.55 (d, 8H, ² J=11.1 Hz), 3.69 (t, 2H), 4.6-5.1(bs, OH), 7.27 (d, 2H, pyridyl-H, ³ J=4.8 Hz), 7.4-7.7 (s, NH), 8.23 (s,2H, pyridyl-H), 8.51 (d, 2H, pyridyl-H, ³ J=4.8 Hz)

¹³ C-NMR (75 MHz, DMSO-d₆, 25° C.): δ=30.7 (CH₂), 59.7 (CH₂ OH), 61.7(CR₄), 63.34 (CR₄), 78.5 (CCO), 122.8, 126.0, 146.0, 148.7, 155.0(pyridine-C, CH₂), 170.2 (CONH)

IR (KBr/solid) [cm⁻¹ ]: 3336, 2936, 2880, 1675, 1597, 1559, 1533, 1465,1363, 1051 (vs)

FAB.sup.⊕ MS (m-NBA, m/e): 833.3, 855.3, 871.3, 965.2 (M+H).sup.⊕,(M+Na).sup.⊕, (M+K).sup.⊕, (M+Cs).sup.⊕

C₃₄ H₅₂ N₆ O₁₈ (832.3)

EXAMPLE 11

Preparation of semicage or cage-like hydrophilic ligands according toreaction scheme III (FIGS. 9a and b) ##STR6## Preparation: 2.6 g (10mmol) bipyridine-methylbromide 80 ml 2-methoxyethylamine 10 g potassiumcarbonate

Procedure:

The bromide was added to a suspension of powderized potassium carbonatein 2-methoxyethylamine while stirring. The suspension was then stirredfor 12 h at room temperature. Subsequently it was filtered, the excess2-methoxyethylamine was removed by distillation and the residue wasdried in a vacuum. The residue was chromatographed (SiO₂ ; CH₂ Cl₂ /CH₃OH/NH₃, 100:10:1).

A light yellow oil was obtained.

Yield: 1.06 g (3.85 mmol) 38%

¹ H-NMR (250 MHz, CDCl₃): 1.95 (s, 1H, NH); 2.26 (s, 3H, pyridyl-CH₃),2.7 (t, ³ J=5.28 Hz, 2H, OCH₂); 3.24 (s, 3H, OCH₃); 3.41 (t, ³ J=5.28Hz, 2H, NCH₂); 3.76 (s, 2H, pyridyl-CH₂), 7.5 (dd, ³ J=8.35 Hz, ⁴ J=2.17Hz, 1H, pyridyl H); 7.69 (dd, ³ J=8.19 Hz, ⁴ J=2.24 Hz, 1H), pyridyl H);8.17 (d, 3J=8.02 Hz, 1H, pyridyl-H); 8.22 (d, ³ J=8.24 Hz, 1H,pyridyl-H); 8.39 (d ⁴ J=1.84 Hz, 1H, pyridyl H); 8.85 (d, ⁴ J=1.85 Hz,1H, pyridyl H) ppm. ##STR7##

Preparation: 2.26 (10 mmol) tri-alcohol 6.67 (35 mmol) tosyl chloride

Procedure

A solution of the tri-alcohol in 20 ml pyridine was slowly admixed undera protecting gas with a solution of tosyl chloride in 20 ml pyridinewhile cooling and stirring so that the temperature of the reactionmixture did not exceed 10° C. Then it was stirred for a further 24 h atroom temperature. Subsequently it was carefully poured onto a mixture of10 ml water, 20 ml methanol and 8 ml concentrated hydrochloric acid. Theprecipitated product and separated oil was filtered or separated andpurified chromatographically (SiO₂ ; CH₂ Cl₂ /CH₃ OH/NH₃, 100:10:1). Oneobtains colourless crystals.

Melting point: 57-59° C.

¹ H-NMR (250 MHz, CD₂ Cl₂): 2.4 (s, 9H, Ar--CH₃); 3.25 (s, 2H, CH₂);3.88 (s, 6H, CH₂); 4.22 (s, 2H, CH₂); 7.02-7.1 (m, 2H, Ar--H); 7.25-7.3(m, 3H, Ar--H); 7.31 (d, ³ J=6.46 Hz, 6H, Ar--H); 7.31 (d, ³ J=6.46 Hz,6H, Ar--H); 7.67 (d, ³ J=6.46 Hz, Ar--H) ppm

¹³ C-NMR and DEPT-135 (62.8 MHz, CDCl₃); 145.36; 137.20; 131.75; 43.82(C_(q)); 130.05; 128.39; 128.33; 127.92; 127.75; 127.23 (CH); 73.28,66.71; 66.31 (CH₂); 21.67 (CH₃) ppm

EXAMPLE 13

Use of hydrophilic metal complexes with a charged linker inimmunological tests

A double-antigen bridge test was carried out to detect specificantibodies against hepatitis C virus (HCV). For this the sample liquidwas incubated with a ruthenium-labelled antigen and a biotinylatedantigen against the antibody to be determined in the presence of a solidphase coated with streptavidin. The presence of anti-HCV antibodies inthe sample liquid was determined by determining the label in the solidphase by electro-chemiluminescence according to the Flash system.

A HCV polypeptide was used as an antigen which contains the amino acids1207-1488 of HCV. The amino acid sequence and the synthesis of such apolypeptide is described in DE-A-44 28 705.4.

In order to derivatize the HCV polypeptide with ruthenium complexesactivated with succinimide ester, the lyophilized polypeptide wasdissolved in a 100 mM sodium phosphate buffer pH 6.5, 0.1% SDS at aprotein concentration of 10 mg/ml. The pH value was set to 8.5 byaddition of 5 M and the solution was supplemented with dithiothreitol toa final concentration of 2 mM. The amount of a ruthenium complexactivated with a succinimide ester in DMSO that corresponds to thedesired offered stoichiometry was added to this solution and it wassubsequently incubated for 60 min at 65° C. while stirring. The reactionwas terminated by supplementing the reaction mixture with lysine to afinal concentration of 10 mM and incubating it for a further 30 min.Subsequently the mixture was dialysed against 100 mM sodium phosphatebuffer pH 6.5, 0.1% SDS. The resulting protein solution was admixed withsucrose (final concentration 6.5% (w/v)) and lyophilized in portions.

For the production of a HCV polypeptide derivatized with a rutheniumcomplex activated with maleinimide, the polypeptide was taken up in 100mM sodium phosphate buffer pH 6.5, 0.1% SDS (protein concentration 10mg/ml). An amount of the maleinimide-activated ruthenium complex in DMSOthat corresponds to the desired offered stoichiometry was added to thissolution and it was incubated for 60 min at 25° C. while stirring. Thereaction was terminated by supplementing the reaction mixture withcysteine to a final concentration of 10 mM and further incubating it for30 min. Afterwards the reaction mixture was dialysed as described above,admixed with sucrose and lyophilized in portions.

Three experiments were carried out in which different ruthenylatedantigens were used each time. For experiment A (comparison) theruthenium complex according to EP-A-0 580 979 used as the startingmaterial in examples 5 and 6 was coupled in a stoichiometric ratio of1:3 to the polypeptide. For experiment B the polypeptide was coupled tothe hydrophilic ruthenium complex according to the invention produced inexample 5 in a stoichiometric ratio of 1:3. For experiment C thepolypeptide was coupled to the hydrophilic ruthenium complex produced inexample 6 in a stoichiometric ratio of 1:1. In all 3 experiments apolypeptide was used as the biotinylated antigen which had been coupledto a maleimide-activated biotin in a stoichiometric ratio of 1:6. Theruthenylated and biotinylated antigens were in each case used at aconcentration of 400 ng/ml test liquid.

The results of experiments A, B and C are shown in Table 2 in ECLcounts. It can be seen that a reliable differentiation between anegative serum sample and a critical positive serum sample can only beachieved by using the hydrophilic metal complexes according to theinvention as marker groups. This is shown by a higher positive/negativeratio.

                  TABLE 4                                                         ______________________________________                                        Experiment   A (comparison)                                                                             B        C                                          ______________________________________                                        negative sample                                                                            323317       84584    44274                                        positive sample 465769 346734 313185                                          Ratio 1.4 4 7                                                                 positive/negative                                                           ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 23                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Asn Asn Thr Arg Lys Ser Ile Ser Ile Gly Pr - #o Gly Arg Ala Phe        Tyr                                                                             1               5   - #                10  - #                15              - - Thr                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Asn Thr Thr Arg Ser Ile Ser Ile Gly Pro Gl - #y Arg Ala Phe Tyr          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Ile Asp Ile Gln Glu Glu Arg Arg Met Arg Il - #e Gly Pro Gly Met Ala      1               5   - #                10  - #                15               - - Trp Tyr Ser                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Le - #u Leu Gly Ile Trp          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Le - #u Lys Asp Gln Gln Leu      1               5   - #                10  - #                15               - - Leu Gly Ile Trp Gly Ala Ser Gly                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Le - #u Ala Leu Glu Thr Leu      1               5   - #                10  - #                15               - - Leu Gln Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Il - #e Cys Thr Thr Ala Val      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Al - #a Val Pro Trp Asn Ala      1               5   - #                10  - #                15               - - Ser Trp Ser                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Se - #r Ser Gly Lys Leu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Ala Leu Glu Thr Leu Leu Gln Asn Gln Leu Le - #u Ser Leu Trp              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cy - #s His Thr Thr              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Gln Ala Gln Leu Asn Ser Trp Gly Cys Ala Ph - #e Arg Gln Val Cys His      1               5   - #                10  - #                15               - - Thr Thr Val Pro Trp Pro Asn Asp Ser Leu Th - #r                                      20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Lys Asn Lys Arg Asn Thr Asn Arg Arg                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gl - #n Asp Val Lys Phe Pro      1               5   - #                10  - #                15               - - Gly Gly Gly Gln Ile Val Gly Gly Val                                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Le - #u Ala Glu Gln Phe Lys      1               5   - #                10  - #                15               - - Gln Gln Ala Leu Gly Leu Leu Gln Thr                                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Ser Arg Gly Asn His Val Ser Pro Thr His Ty - #r Val Pro Glu Ser Asp      1               5   - #                10  - #                15               - - Ala Ala                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Tr - #p Ala Arg Pro Asp          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gl - #n Asp Val Lys Phe Pro      1               5   - #                10  - #                15               - - Gly Gly Gly Gln Ile Val Gly Gly Val Leu Le - #u Pro Arg Arg                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg As - #n Thr Asn Arg Arg          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pr - #o Arg Arg Gly Pro Arg      1               5   - #                10  - #                15               - - Leu Gly                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gl - #n Ile Val Gly Gly Val      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Ser Arg Gly Asn His Val Ser Pro Thr His Ty - #r Val Pro Glu Ser Asp      1               5   - #                10  - #                15               - - Ala Ala                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Ser Gln His Leu Pro Tyr Ile Glu Gln                                      1               5                                                            __________________________________________________________________________

We claim:
 1. A metal complex having the formula:

    [M(L.sub.1 L.sub.2 L.sub.3)].sub.n --X.sub.m A

wherein M is a divalent or trivalent metal cation selected from thegroup consisting of a rare earth metal cation and a transition metalcation; L₁, L₂ and L₃ are the same or different, and each is a ligandhaving at least two nitrogen-containing heterocyclic rings, wherein L₁,L₂ and L₃ are bound to the metal cation via nitrogen atoms; X is areactive or activatable functional group selected from the groupconsisting of an activated carboxylic acid group, a maleimide, acarboxylic acid, a thiol, a halide and a photoactivatable group; n is 1to 10; m is 1 to 6; and A is at least one negatively charged group,wherein the metal complex contains at least one hydrophilic groupselected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂-C₃ alkylenethio group, a C₂ -C₃ alkyleneamino group and a polyhydroxygroup, wherein the at least one hydrophilic group is located at at leastone of the following locations (a) and (b):(a) as a substituent orcomponent of a substituent of one of the ligands L₁, L₂ and L₃ ; and (b)as a linker or component of a linker which links the group X and one ofthe ligands L₁, L₂ and L₃.
 2. The metal complex as claimed in claim 1,wherein M is selected from the group consisting of ruthenium ion,rhenium ion, osmium ion, chromium ion and iridium ion.
 3. The metalcomplex as claimed in claim 1, wherein M is a ruthenium ion.
 4. Themetal complex as claimed in claim 1, wherein each of L₁, L₂ and L₃independently contains a ring system selected from the group consistingof bipyridine and phenanthroline.
 5. The metal complex as claimed inclaim 1, wherein A is selected from the group consisting of ahexafluorophosphate group, a trifluoroacetate group, a tetrafluoroborategroup and a halide ion.
 6. The metal complex as claimed in claim 1,wherein the polyhydroxy group is a group of formula (IIa) or (IIb)

    --NR--W                                                    (IIa)

    --O--W                                                     (IIb)

wherein W is an organic residue having at least two hydroxy groups and Ris hydrogen or C₁ -C₅ alkyl.
 7. The metal complex as claimed in claim 6,wherein the polyhydroxy group has a formula --NR--C(CH₂ OH)₃, wherein Ris as defined in claim
 6. 8. The metal complex as claimed in claim 1,wherein the C₂ -C₃ alkyleneoxy group is an ethyleneoxy group, the C₂ -C₃alkylenethio group is an ethylenethio group, and the C₂ -C₃alkyleneamino group is an ethyleneamino group.
 9. The metal complex asclaimed in claim 1, wherein the metal complex contains 1-30 hydrophilicgroups each independently selected from the group consisting of a C₂ -C₃alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃alkyleneamino group.
 10. The metal complex as claimed in claim 1,wherein the metal complex is of formula (III): ##STR8## wherein M, X andA are as defined in claim 1;one of R₁ through R₆ is bound to --X; andthe remainder of R₁ through R₆ which are not bound to --X are eachindependently selected from the group consisting of hydrogen, C₁ -C₅alkyl and a substituent containing at least one hydrophilic group whichis selected from the group consisting of a C₂ -C₃ alkyleneoxy group, aC₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group, wherein atleast one of the remainder of R₁ through R₆ is the substituentcontaining the at least one hydrophilic group.
 11. The metal complex asclaimed in claim 1, wherein the metal complex is of formula (IIIa):##STR9## wherein M, X and A are as defined in claim 1;R₁ through R₅ areeach independently selected from the group consisting of hydrogen, C₁-C₅ alkyl and a substituent containing at least one hydrophilic groupwhich is selected from the group consisting of a C₂ -C₃ alkyleneoxygroup, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group; sis 0-6; and Y is a linker having 1-10 hydrophilic groups eachindependently selected from the group consisting of a C₂ -C₃ alkyleneoxygroup, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group. 12.The metal complex as claimed in claim 1, wherein the metal complex is offormula (IIIb): ##STR10## wherein M, X and A are as defined in claim1;R₁ through R₅ are each independently selected from the groupconsisting of hydrogen, C₁ -C₅ alkyl and a substituent containing 1-10hydrophilic groups each of which is selected from the group consistingof a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃alkyleneamino group, wherein at least one of R₁ through R₅ is thesubstituent containing the 1-10 hydrophilic groups; and s is 0-6. 13.The metal complex as claimed in claim 1, wherein the metal complex is offormula (IV): ##STR11## wherein M, Y, X and n are as defined in claim1;R₁ through R₃ are each independently selected from the groupconsisting of hydrogen, C₁ -C₅ alkyl and a substituent containing atleast one hydrophilic group which is selected from the group consistingof a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃alkyleneamino group; and Y is a linker having 1-10 hydrophilic groupseach independently selected from the group consisting of a C₂ -C₃alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃alkyleneamino group.
 14. The metal complex as claimed in claim 1,wherein the metal complex is of formula (V): ##STR12## wherein M, X andA are as defined in claim 1;Z is a linker; R₁ ' through R₄ ' are eachindependently selected from the group consisting of hydrogen, C₁ -C₅alkyl and a substituent containing at least one polyhydroxy group,wherein at least one of R₁ ' through R₄ ' is the substituent containingthe at least one polyhydroxy group; and R₅ ' is hydrogen or C₁ -C₅alkyl.
 15. The metal complex as claimed in claim 14, wherein the --OHgroup of the at least one polyhydroxy group is substituted by ahydrophilic group.
 16. The metal complex as claimed in claim 15, whereinthe hydrophilic group comprises an element of formula (VIa) or (VIb):

    --A.sub.1 --NR--W.sub.1 (A.sub.2 --NR--W.sub.2).sub.n '    (VIa)

    --A.sub.1 --O--W.sub.1 (A.sub.2 --O--W.sub.2).sub.n '      (VIb)

wherein A₁ and A₂ are each a same or different linker; W₁ and W₂ areeach a same or different organic residue having at least two hydroxygroups; R is hydrogen or C₁ -C₅ alkyl; and n' is 0 or the same number asthe number of hydroxy groups on W₁.
 17. A conjugate, comprising a metalcomplex coupled with a biological substance, wherein the metal complexhas the formula:

    [M(L.sub.1 L.sub.2 L.sub.3)].sub.n --X.sub.m A

wherein M is a divalent or trivalent metal cation selected from thegroup consisting of a rare earth metal cation and a transition metalcation; L₁, L₂ and L₃ are the same or different, and each is a ligandhaving at least two nitrogen-containing heterocyclic rings, wherein L₁,L₂ and L₃ are bound to the metal cation via nitrogen atoms; X is areactive or activatable functional group; n is 1 to 10; m is 1 to 6; andA is at least one negatively charged group, wherein the metal complexcontains at least one hydrophilic group selected from the groupconsisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group, aC₂ -C₃ alkyleneamino group and a polyhydroxy group, wherein the at leastone hydrophilic group is located at at least one of the followinglocations (a) and (b):(a) as a substituent or component of a substituentof one of the ligands L₁, L₂ and L₃ ; and (b) as a linker or componentof a linker which links the group X and one of the ligands L₁, L₂ andL₃.
 18. The conjugate as claimed in claim 17, wherein the biologicalsubstance is selected from the group consisting of biotin, an antibody,an antibody fragment, a nucleic acid, a polypeptide antigen, animmunologically reactive peptide and a hapten.